The recent deciphering of entire genomic sequences of different organisms, including humans, has resulted in a demand to decipher three-dimensional structures of protein gene products. Determining the structures of proteins may allow researchers to compile structural information that will facilitate predictions of function for almost any protein from knowing its coding sequences. Gaining a better understanding of protein structure and function may enable drug researchers to develop new drug treatments that target specific human, animal, and plant diseases. The human body alone has an estimated 52,000 different proteins. Determining the structures to atomic resolution for all these proteins is a daunting challenge, at best. X-ray crystallography currently offers one method to achieve this goal and is the only method to date for determining macromolecules greater than 35,000 Daltons.
Today, advanced recombinant DNA methods, systematic approaches for protein crystallization, and highly developed X-ray diffraction instruments and procedures contribute to determining protein structure. The limiting step in protein structural determination is the ability to obtain protein crystals that are suitable for X-ray diffraction. Suitable crystals should be able to diffract to atomic resolutions greater than 3 Angstroms with reflections that can be readily indexed.
The process for obtaining crystals suitable for X-ray diffraction normally is divided into four discrete steps. The first step includes determining conditions for initial protein crystallization. There are numerous factors influencing crystal formation, which include: pH, ionic strength, temperature, gravity, and viscosity, to name but a few. Second, the initial crystallization conditions are optimized to produce crystals that are suitable for X-ray diffraction. This step entails making minute adjustments to the many crystallization parameters to produce the highest quality crystal.
In the third step, the crystals are treated with a cryoprotectant solution so that the protein crystal will tolerate supercooled conditions. Protein crystals are very sensitive to X-ray radiation and therefore data collection must be performed under super cooled conditions. During this step, the researcher typically tests the crystal in a variety of cryogenic solutions at different concentrations and soak times.
In the final step, strong X-ray scattering atoms are required for ab initio phasing. Atoms such as sulfur or metal ions are intrinsic to most proteins and are often used for crystallographic phasing by using the atoms' anomalous signals. However, halides or heavy metals provide much higher X-ray scattering signals for effective phasing and they are typically incorporated into the protein in an invasive manner. This step usually requires rigorous testing to find appropriate scattering atoms that can isomorphously incorporate into the crystal without damaging the crystalline order.
Typically, the third and fourth steps require the researcher to manually transfer protein crystals between different solutions followed by mounting the crystal on cryoloops for X-ray analysis. These steps require that the researcher delicately handle the crystal because any over excursion of force or mishandling could damage the crystal. Current methods for growing and analyzing protein crystals are time intensive, often fail to produce useful crystals, and require that the researcher use extreme care when handling the crystals.
From the foregoing, it should be readily apparent that obtaining protein crystals and their subsequent preparation for X-ray analysis is a very time consuming and limiting step in determining protein structure. Consequently, more efficient methods are needed for growing protein crystals suitable for analysis.